Human DNA Polymerase ? (Pol?) is essential for many cellular processes including replication of the leading strand during DNA synthesis. Unlike Polymerase ? (Pol?), the lagging strand polymerase, Pol? is thought to replicate long stretches of DNA continuously and thus should be highly processive. Upon encountering an unrepaired lesion in the DNA template, it may also be necessary for Pol? to dissociate or be displaced to prevent collapse of the replication fork. Proliferating cell nuclear antigen (PCNA) is a ring-shaped clamp that acts as a polymerase processivity factor and recruits specialized translesion synthesis (TLS) polymerases that can bypass DNA lesions. I propose to characterize the interactions of PCNA and Pol? to determine if they form a stable complex as has been observed for the Pol? holoenzyme. This will be done with surface plasmon resonance to measure binding affinities and a FRET-based assay to monitor holoenzyme assembly. I will then characterize the stability and processivity of Pol? both with and without PCNA. DNA synthesis by Pol? will be assessed with ensemble assays and using single molecule techniques such as magnetic tweezers. I will then explore the mechanism of polymerase exchange at sites of DNA damage using Pol? and the TLS Polymerase ? (Pol?) along with ubiquitinated PCNA. Ubiquitination of PCNA by Rad6 and Rad18 is recognized as an important signal for the recruitment of a TLS polymerase, but the effect of this modification on Pol? holoenzyme stability and processivity have not been assessed. I will determine the mechanism of polymerase exchange between Pol? and Pol? using ensemble assays. While this will initially be done by loading PCNA that is already ubiquitinated, a further goal of this projec will be to delineate the requirements for PCNA ubiquitination at a site of damage. This will be done first by monitoring PCNA ubiquitination on different templates, and in the presence of various components of the replisome such as RPA, Pol?, Pol?, or Pol?. FRET-based assays, using fluorescently labeled DNA or protein, will reveal what proteins are necessary for recruitment and activity of Rad6/Rad18. Together these studies will yield a more detailed understanding of the functioning of the human leading strand polymerase during both normal DNA replication and upon encountering a damaged template. PCNA is likely integral for both of these processes, but the regulation of its posttranslational modification and functional consequences remain to be fully characterized.